Journal: Microbiology Spectrum

Article Title: Rapid Point-of-Care Tests Using Staphylococcal Protein A Can Detect Early IgM Responses in HIV-1 and Treponema pallidum Infections

doi: 10.1128/spectrum.03309-22

Figure Lengend Snippet: Antibody detection in HIV-1 seroconversion panel PRB-927 and effect of sample pretreatment with 0.1 M 2-ME. (A) Micro Reader reflectance values generated with the DPP HIV 1/2 (red lines) and DPP HIV 1/2 IgM assays (blue lines) are shown for samples tested before (solid lines) and after 2-ME treatment (dashed lines). Framed are test results for samples 03 and 04 collected during initial IgM seroconversion (days 33 and 35 since 1st bleed), in which antibody detection by each of the two immunoassays was diminished after 2-ME treatment by at least 50% signal reduction. (B) Results show differentially evolving susceptibility to the 2-ME treatment of postseroconversion samples tested by the DPP HIV 1/2 (red line) and DPP HIV 1/2 IgM assays (blue line). Framed is the same data subset as that included in panel A but results are shown in percent signal reduction over time.

Article Snippet: DPP HIV 1/2 and DPP HIV-Syphilis tests were converted into investigational IgM-detecting assays, named DPP HIV 1/2 IgM and DPP HIV-Syphilis IgM, respectively, by replacing SpA coupled to gold nanoparticles with mouse monoclonal antibody to human IgM (HyTest Ltd., Turku, Finland), a specific reagent for IgM detection validated in commercial DPP products ( , ).

Techniques: Generated

Journal: Microbiology Spectrum

Article Title: Rapid Point-of-Care Tests Using Staphylococcal Protein A Can Detect Early IgM Responses in HIV-1 and Treponema pallidum Infections

doi: 10.1128/spectrum.03309-22

Figure Lengend Snippet: IgM antibody detection by SpA-based rapid point-of-care tests in HIV-1 seroconversion panels ^a

Article Snippet: DPP HIV 1/2 and DPP HIV-Syphilis tests were converted into investigational IgM-detecting assays, named DPP HIV 1/2 IgM and DPP HIV-Syphilis IgM, respectively, by replacing SpA coupled to gold nanoparticles with mouse monoclonal antibody to human IgM (HyTest Ltd., Turku, Finland), a specific reagent for IgM detection validated in commercial DPP products ( , ).

Techniques:

Journal: Microbiology Spectrum

Article Title: Rapid Point-of-Care Tests Using Staphylococcal Protein A Can Detect Early IgM Responses in HIV-1 and Treponema pallidum Infections

doi: 10.1128/spectrum.03309-22

Figure Lengend Snippet: Antibody detection in AccuVert Syphilis Seroconversion Panel PSS901 and effect of sample pre-treatment with 0.1 M 2-ME. (A) Micro Reader reflectance values generated with the DPP HIV-Syphilis (red lines) and DPP HIV-Syphilis IgM assays (blue lines) are shown for samples tested before (solid lines) and after 2-ME treatment (dashed lines). Framed are test results for samples 06 to 09 collected during the initial IgM response (days 45 to 59 since 1st bleed), in which antibody detection with each of the two immunoassays was diminished after 2-ME treatment by at least 50% signal reduction. (B) Results show differentially evolving susceptibility to the 2-ME treatment of postseroconversion samples (same as framed in panel A) tested by the DPP HIV-Syphilis (red line) and DPP HIV-Syphilis IgM assays (blue line).

Article Snippet: DPP HIV 1/2 and DPP HIV-Syphilis tests were converted into investigational IgM-detecting assays, named DPP HIV 1/2 IgM and DPP HIV-Syphilis IgM, respectively, by replacing SpA coupled to gold nanoparticles with mouse monoclonal antibody to human IgM (HyTest Ltd., Turku, Finland), a specific reagent for IgM detection validated in commercial DPP products ( , ).

Techniques: Generated

Journal: Microbiology Spectrum

Article Title: Rapid Point-of-Care Tests Using Staphylococcal Protein A Can Detect Early IgM Responses in HIV-1 and Treponema pallidum Infections

doi: 10.1128/spectrum.03309-22

Figure Lengend Snippet: IgM antibody detection by DPP HIV-Syphilis assay in active syphilis ^a

Article Snippet: DPP HIV 1/2 and DPP HIV-Syphilis tests were converted into investigational IgM-detecting assays, named DPP HIV 1/2 IgM and DPP HIV-Syphilis IgM, respectively, by replacing SpA coupled to gold nanoparticles with mouse monoclonal antibody to human IgM (HyTest Ltd., Turku, Finland), a specific reagent for IgM detection validated in commercial DPP products ( , ).

Techniques:

Journal: Microbiology Spectrum

Article Title: Rapid Point-of-Care Tests Using Staphylococcal Protein A Can Detect Early IgM Responses in HIV-1 and Treponema pallidum Infections

doi: 10.1128/spectrum.03309-22

Figure Lengend Snippet: Correlation between antibody levels detected by SpA or anti-IgM reagent in early HIV-1 seroconversion and syphilis samples. (A) Micro Reader reflectance values generated with the DPP HIV-1/2 and DPP HIV-1/2 IgM assays are shown for the HIV-1 seroconversion samples included in Table 1 , Groups 2 and 3 (circles), and matching preseroconversion members (triangles). (B) Micro Reader reflectance values generated for treponemal antibody levels with the DPP HIV-Syphilis and DPP HIV-Syphilis IgM assays are shown for the syphilis-positive samples listed in Table 2 (circles) and preseroconversion panel members (triangles). Horizontal and vertical dashed lines indicate cutoff values used for test interpretation: DPP HIV-1/2, 10 RLU; DPP HIV-1/2 IgM, 40 RLU; DPP HIV-Syphilis, 7 RLU; DPP HIV-Syphilis IgM, 60 RLU.

Article Snippet: DPP HIV 1/2 and DPP HIV-Syphilis tests were converted into investigational IgM-detecting assays, named DPP HIV 1/2 IgM and DPP HIV-Syphilis IgM, respectively, by replacing SpA coupled to gold nanoparticles with mouse monoclonal antibody to human IgM (HyTest Ltd., Turku, Finland), a specific reagent for IgM detection validated in commercial DPP products ( , ).

Techniques: Generated

Journal: International Journal of Infectious Diseases

Article Title: Clinical evaluation of commercial automated SARS-CoV-2 immunoassays

doi: 10.1016/j.ijid.2020.12.003

Figure Lengend Snippet: Overview of suppliers. Chemiluminescence immunoassay (CLIA), enzyme-linked immunosorbent assay (ELISA), nucleocapsid protein (N-protein), spike protein (S-protein).

Article Snippet: The most sensitive SARS-CoV-2 antibody test was the Euroimmun IgA test with 87%, followed by the Epitope Diagnostics IgG test with 83%, the Shenzhen YHLO Biotech IgG test with 77%, the Roche IgM/IgG test with 77%, the Euroimmun IgG test with 75%, the Diasorin IgG test with 53%, the Epitope Diagnostics IgM test with 52%, the Snibe IgG Test with 47%, the Shenzhen YHLO Biotech IgM test with 35%, and the Snibe IgM test with 26%.

Techniques: Chemiluminescence Immunoassay, Enzyme-linked Immunosorbent Assay, Diagnostic Assay

Journal: International Journal of Infectious Diseases

Article Title: Clinical evaluation of commercial automated SARS-CoV-2 immunoassays

doi: 10.1016/j.ijid.2020.12.003

Figure Lengend Snippet: Precision box-whiskers plot of the inter-assay and intra-assay variations in the IgM and IgG SARS-CoV-2 assay from Shenzhen YHLO Biotech and Roche Elecsys Anti-SARS-CoV-2 ECLIA.

Article Snippet: The most sensitive SARS-CoV-2 antibody test was the Euroimmun IgA test with 87%, followed by the Epitope Diagnostics IgG test with 83%, the Shenzhen YHLO Biotech IgG test with 77%, the Roche IgM/IgG test with 77%, the Euroimmun IgG test with 75%, the Diasorin IgG test with 53%, the Epitope Diagnostics IgM test with 52%, the Snibe IgG Test with 47%, the Shenzhen YHLO Biotech IgM test with 35%, and the Snibe IgM test with 26%.

Techniques: Inter Assay, Intra Assay

Journal: International Journal of Infectious Diseases

Article Title: Clinical evaluation of commercial automated SARS-CoV-2 immunoassays

doi: 10.1016/j.ijid.2020.12.003

Figure Lengend Snippet: Z-Score comparison. Comparison between the IgM and IgG SARS-CoV-2 assay from Shenzhen YHLO Biotech and the IgM/IgG assay by Roche Diagnostics. Data were transformed to Z-scores, as described in the materials and methods section, and the cut-off values were uniformly set to log (1). Measurement of qRT-PCR-confirmed COVID-19 cases (n = 59) and pre-pandemic negative controls (n = 50).

Article Snippet: The most sensitive SARS-CoV-2 antibody test was the Euroimmun IgA test with 87%, followed by the Epitope Diagnostics IgG test with 83%, the Shenzhen YHLO Biotech IgG test with 77%, the Roche IgM/IgG test with 77%, the Euroimmun IgG test with 75%, the Diasorin IgG test with 53%, the Epitope Diagnostics IgM test with 52%, the Snibe IgG Test with 47%, the Shenzhen YHLO Biotech IgM test with 35%, and the Snibe IgM test with 26%.

Techniques: Transformation Assay, Quantitative RT-PCR

Journal: International Journal of Infectious Diseases

Article Title: Clinical evaluation of commercial automated SARS-CoV-2 immunoassays

doi: 10.1016/j.ijid.2020.12.003

Figure Lengend Snippet: AUROC curves. AUROC graphs for the highest ((A) Shenzhen YHLO Biotech IgG, AUC: 0.97) and lowest ((B) Snibe IgM, AUC: 0.66) values; detailed information can be found in .

Article Snippet: The most sensitive SARS-CoV-2 antibody test was the Euroimmun IgA test with 87%, followed by the Epitope Diagnostics IgG test with 83%, the Shenzhen YHLO Biotech IgG test with 77%, the Roche IgM/IgG test with 77%, the Euroimmun IgG test with 75%, the Diasorin IgG test with 53%, the Epitope Diagnostics IgM test with 52%, the Snibe IgG Test with 47%, the Shenzhen YHLO Biotech IgM test with 35%, and the Snibe IgM test with 26%.

Techniques:

Journal: International Journal of Infectious Diseases

Article Title: Clinical evaluation of commercial automated SARS-CoV-2 immunoassays

doi: 10.1016/j.ijid.2020.12.003

Figure Lengend Snippet: AUROC calculations. Diagnostic performance of all immunosorbent assays listed in Table 2 , as calculated by AUROC values.

Article Snippet: The most sensitive SARS-CoV-2 antibody test was the Euroimmun IgA test with 87%, followed by the Epitope Diagnostics IgG test with 83%, the Shenzhen YHLO Biotech IgG test with 77%, the Roche IgM/IgG test with 77%, the Euroimmun IgG test with 75%, the Diasorin IgG test with 53%, the Epitope Diagnostics IgM test with 52%, the Snibe IgG Test with 47%, the Shenzhen YHLO Biotech IgM test with 35%, and the Snibe IgM test with 26%.

Techniques: Diagnostic Assay

Journal: International Journal of Infectious Diseases

Article Title: Clinical evaluation of commercial automated SARS-CoV-2 immunoassays

doi: 10.1016/j.ijid.2020.12.003

Figure Lengend Snippet: Longitudinal monitoring. Kinetics of antibody concentrations were analysed for 15 patients with qRT-PCR-confirmed COVID-19 infections. Multiple blood samplings were performed for up to 14 weeks after qRT-PCR. The cut-off values for the Shenzhen YHLO Biotech assays (10 AU/mL) are indicated with horizontal lines. The courses of antibody concentrations were grouped into four categories: (I) decreasing (blue), (II) increasing (orange), (III) steady (green), and (IV) variable (yellow). Most interestingly, antibody formation could not be detected for IgM or IgG in one case, even 9 weeks after a positive qRT-PCR result (triangle symbols, Patient 1).

Article Snippet: The most sensitive SARS-CoV-2 antibody test was the Euroimmun IgA test with 87%, followed by the Epitope Diagnostics IgG test with 83%, the Shenzhen YHLO Biotech IgG test with 77%, the Roche IgM/IgG test with 77%, the Euroimmun IgG test with 75%, the Diasorin IgG test with 53%, the Epitope Diagnostics IgM test with 52%, the Snibe IgG Test with 47%, the Shenzhen YHLO Biotech IgM test with 35%, and the Snibe IgM test with 26%.

Techniques: Quantitative RT-PCR

Journal: International Journal of Infectious Diseases

Article Title: Clinical evaluation of commercial automated SARS-CoV-2 immunoassays

doi: 10.1016/j.ijid.2020.12.003

Figure Lengend Snippet: Overview of suppliers. Chemiluminescence immunoassay (CLIA), enzyme-linked immunosorbent assay (ELISA), nucleocapsid protein (N-protein), spike protein (S-protein).

Article Snippet: Overall diagnostic sensitivities were: Euroimmun (IgA) 87%; Epitope Diagnostics (IgG) 83%; YHLO (IgG) 77%; Roche (IgM/IgG) 77%; Euroimmun (IgG) 75%; Diasorin (IgG) 53%; Epitope Diagnostics (IgM) 52%; Snibe (IgG) 47%; YHLO (IgM) 35%; and Snibe (IgM) 26%.

Techniques: Chemiluminescence Immunoassay, Enzyme-linked Immunosorbent Assay, Diagnostic Assay

Journal: International Journal of Infectious Diseases

Article Title: Clinical evaluation of commercial automated SARS-CoV-2 immunoassays

doi: 10.1016/j.ijid.2020.12.003

Figure Lengend Snippet: Precision box-whiskers plot of the inter-assay and intra-assay variations in the IgM and IgG SARS-CoV-2 assay from Shenzhen YHLO Biotech and Roche Elecsys Anti-SARS-CoV-2 ECLIA.

Article Snippet: Overall diagnostic sensitivities were: Euroimmun (IgA) 87%; Epitope Diagnostics (IgG) 83%; YHLO (IgG) 77%; Roche (IgM/IgG) 77%; Euroimmun (IgG) 75%; Diasorin (IgG) 53%; Epitope Diagnostics (IgM) 52%; Snibe (IgG) 47%; YHLO (IgM) 35%; and Snibe (IgM) 26%.

Techniques: Inter Assay, Intra Assay

Journal: International Journal of Infectious Diseases

Article Title: Clinical evaluation of commercial automated SARS-CoV-2 immunoassays

doi: 10.1016/j.ijid.2020.12.003

Figure Lengend Snippet: Z-Score comparison. Comparison between the IgM and IgG SARS-CoV-2 assay from Shenzhen YHLO Biotech and the IgM/IgG assay by Roche Diagnostics. Data were transformed to Z-scores, as described in the materials and methods section, and the cut-off values were uniformly set to log (1). Measurement of qRT-PCR-confirmed COVID-19 cases (n = 59) and pre-pandemic negative controls (n = 50).

Article Snippet: Overall diagnostic sensitivities were: Euroimmun (IgA) 87%; Epitope Diagnostics (IgG) 83%; YHLO (IgG) 77%; Roche (IgM/IgG) 77%; Euroimmun (IgG) 75%; Diasorin (IgG) 53%; Epitope Diagnostics (IgM) 52%; Snibe (IgG) 47%; YHLO (IgM) 35%; and Snibe (IgM) 26%.

Techniques: Transformation Assay, Quantitative RT-PCR

Journal: International Journal of Infectious Diseases

Article Title: Clinical evaluation of commercial automated SARS-CoV-2 immunoassays

doi: 10.1016/j.ijid.2020.12.003

Figure Lengend Snippet: AUROC curves. AUROC graphs for the highest ((A) Shenzhen YHLO Biotech IgG, AUC: 0.97) and lowest ((B) Snibe IgM, AUC: 0.66) values; detailed information can be found in .

Article Snippet: Overall diagnostic sensitivities were: Euroimmun (IgA) 87%; Epitope Diagnostics (IgG) 83%; YHLO (IgG) 77%; Roche (IgM/IgG) 77%; Euroimmun (IgG) 75%; Diasorin (IgG) 53%; Epitope Diagnostics (IgM) 52%; Snibe (IgG) 47%; YHLO (IgM) 35%; and Snibe (IgM) 26%.

Techniques:

Journal: International Journal of Infectious Diseases

Article Title: Clinical evaluation of commercial automated SARS-CoV-2 immunoassays

doi: 10.1016/j.ijid.2020.12.003

Figure Lengend Snippet: Longitudinal monitoring. Kinetics of antibody concentrations were analysed for 15 patients with qRT-PCR-confirmed COVID-19 infections. Multiple blood samplings were performed for up to 14 weeks after qRT-PCR. The cut-off values for the Shenzhen YHLO Biotech assays (10 AU/mL) are indicated with horizontal lines. The courses of antibody concentrations were grouped into four categories: (I) decreasing (blue), (II) increasing (orange), (III) steady (green), and (IV) variable (yellow). Most interestingly, antibody formation could not be detected for IgM or IgG in one case, even 9 weeks after a positive qRT-PCR result (triangle symbols, Patient 1).

Article Snippet: Overall diagnostic sensitivities were: Euroimmun (IgA) 87%; Epitope Diagnostics (IgG) 83%; YHLO (IgG) 77%; Roche (IgM/IgG) 77%; Euroimmun (IgG) 75%; Diasorin (IgG) 53%; Epitope Diagnostics (IgM) 52%; Snibe (IgG) 47%; YHLO (IgM) 35%; and Snibe (IgM) 26%.

Techniques: Quantitative RT-PCR

Journal:

Article Title: The B Lymphocyte-Specific Coactivator BOB.1/OBF.1 Is Required at Multiple Stages of B-Cell Development

doi: 10.1128/MCB.21.5.1531-1539.2001

Figure Lengend Snippet: BOB.1/OBF.1-deficient mice show defects in early B-cell development. (A) Flow cytometry analysis of gated lymphocytes from spleens of 1-week-old control (WT) and BOB.1/OBF.1-deficient (bob.1/obf.1−/−) mice. Cells were stained with anti-IgM-PE and anti-IgD-FITC antibodies or with anti-B220-FITC and anti-PB493-biotin antibodies. The percentages of different B-cell populations are shown in the FACS quadrents. At the bottom, statistical analyses from at least three independent measurements are shown. (B) Flow cytometry analysis of bone marrow lymphocytes from 6- to 8-week-old control (WT) and BOB.1/OBF.1-deficient mice, which were stained with anti-B220-FITC and anti-IgM-PE antibodies. Percentages of pro- and pre-B cells (B220lo IgM−), immature B cells (B220lo IgM+), T1 B cells (B220lo IgMhi), and recirculating B cells (B220hi IgM+) are indicated in the figure. Statistical analyses from 13 independent measurements are shown in Fig. ​Fig.4B4B and C. (C) Level of annexin V on the surface of control (WT) or BOB.1-deficient B220lo bone marrow B cells measured with an anti-annexin V-FITC antibody by flow cytometry. The cells were also stained with an anti-B220-PE antibody. The percentage of annexin V-positive cells is denoted in boldfaced numerals. Statistical analyses from three independent measurements are shown in Fig. ​Fig.4E.4E. (D) Percentage of BrdU-positive lymphocytes in bone marrow and spleen of control (grey bars) and BOB.1/OBF.1-deficient (black bars) mice. Labeling was performed for 3 or 5 days as indicated. Labeled cells were analyzed by flow cytometry after staining with anti-BrdU-FITC antibodies and anti-B220-PE for B cells.

Article Snippet: Cell surface markers were stained using the following reagents: Fc-block (Pharmingen), anti-IgM-phycoerythrin (PE) (Dianova), anti-IgD-fluorescein isothiocyanate (FITC) (Pharmingen), anti-B220-FITC (Pharmingen), monoclonal antibody (MAb) 493-biotin ( 25 ), annexin V (Pharmingen), anti-IgM a -biotin (Pharmingen), anti-IgM b -FITC (Pharmingen), anti-Thy1.1-FITC (Pharmingen), anti-Thy1.2-PE (Pharmingen), and streptavidin-PE (Pharmingen).

Techniques: Flow Cytometry, Staining, Labeling

Journal:

Article Title: The B Lymphocyte-Specific Coactivator BOB.1/OBF.1 Is Required at Multiple Stages of B-Cell Development

doi: 10.1128/MCB.21.5.1531-1539.2001

Figure Lengend Snippet: Rescue of the early B-cell phenotype by conditional expression of transgenic BOB.1/OBF.1. (A) Flow cytometry analysis of bone marrow cells from 6- to 8-week-old dTg BOB.1/OBF.1-deficient mice (dTg-bob.1−/−) stained with anti-B220-FITC and anti-IgM-PE antibodies. Percentages of pro- and pre-B cells (B220lo IgM−), immature B cells (B220lo IgM+), T1 B cells (B220lo IgMhi), and recirculating B cells (B220hi IgM+) are indicated. Transgene expression was turned off by administration of 2 mg of doxycycline (+dox)/ml to the drinking water for 5 days. (B and C) Statistical analysis of the percentages of various B-cell populations from at least four independent mice in each group. (D) Representative analysis of annexin V-positive B cells in bone marrow of mice with the indicated genotypes and treatments. The levels of annexin V on the surface B220+ bone marrow B cells of control mice, BOB.1/OBF.1-deficient mice, dTg BOB.1/OBF.1-deficient mice not treated with doxycycline, and dTg BOB.1/OBF.1-deficient mice treated with doxycycline (left to right) were measured with an anti-annexin V-FITC antibody by flow cytometry. Doxycycline had no effect on the percentage of annexin V staining cells in wild-type or BOB.1/OBF.1-deficient animals not bearing the tetracycline-regulated transgene. (E) Percentage of annexin V-positive bone marrow B cells. Data are shown as averages (± standard deviations) from three independent animals for each genotype.

Article Snippet: Cell surface markers were stained using the following reagents: Fc-block (Pharmingen), anti-IgM-phycoerythrin (PE) (Dianova), anti-IgD-fluorescein isothiocyanate (FITC) (Pharmingen), anti-B220-FITC (Pharmingen), monoclonal antibody (MAb) 493-biotin ( 25 ), annexin V (Pharmingen), anti-IgM a -biotin (Pharmingen), anti-IgM b -FITC (Pharmingen), anti-Thy1.1-FITC (Pharmingen), anti-Thy1.2-PE (Pharmingen), and streptavidin-PE (Pharmingen).

Techniques: Expressing, Transgenic Assay, Flow Cytometry, Staining

Journal:

Article Title: The B Lymphocyte-Specific Coactivator BOB.1/OBF.1 Is Required at Multiple Stages of B-Cell Development

doi: 10.1128/MCB.21.5.1531-1539.2001

Figure Lengend Snippet: Dramatic block of BOB.1-deficient B-cell development in the presence of wild-type cells. (A and B) Flow cytometry analysis of splenic lymphocytes 4 to 8 weeks after the transfer with bone marrow cells from wild-type C57BL/6a and control C57BL/6 mice (wt-a/wt-b) or wild-type C57BL/6a and BOB.1-deficient C57BL/6 mice (wt-a/ko-b). (A) The T-cell populations (T-cell-receptor-positive cells) in the spleen were analyzed with Thy1 antibodies recognizing the two alleles (anti-Thy1.1 and anti-Thy1.2). (B) The contributions of the two allotypes to the splenic B-cell population (B220-positive cells) were estimated by measuring the expression of the alleles for IgM (anti-IgMa and anti-IgMb). Statistical analyses from two independent transfers are shown on the right. (C) Analysis of B-cell populations in the bone marrow using the same antibodies as used for panel B. The diagram on right shows the statistical analysis of both transfers.

Article Snippet: Cell surface markers were stained using the following reagents: Fc-block (Pharmingen), anti-IgM-phycoerythrin (PE) (Dianova), anti-IgD-fluorescein isothiocyanate (FITC) (Pharmingen), anti-B220-FITC (Pharmingen), monoclonal antibody (MAb) 493-biotin ( 25 ), annexin V (Pharmingen), anti-IgM a -biotin (Pharmingen), anti-IgM b -FITC (Pharmingen), anti-Thy1.1-FITC (Pharmingen), anti-Thy1.2-PE (Pharmingen), and streptavidin-PE (Pharmingen).

Techniques: Blocking Assay, Flow Cytometry, Expressing

Journal:

Article Title: The B Lymphocyte-Specific Coactivator BOB.1/OBF.1 Is Required at Multiple Stages of B-Cell Development

doi: 10.1128/MCB.21.5.1531-1539.2001

Figure Lengend Snippet: Conditional correction in the number of peripheral B cells in spleen. Flow cytometry analysis of splenocytes from 6- to 8-week-old control, BOB.1/OBF.1-deficient (bob.1/obf.1−/−) and dTg BOB.1/OBF.1-deficient (dTg-bob.1−/−) mice. Cells were stained in a combination of anti-B220-PE, anti-IgM-biotin, and anti-IgD-FITC antibodies. The percentage of different B-cell populations is denoted by boldfaced numerals. Transgene expression was turned off by administration of 2 mg/of doxycycline (+dox.)/ml to the animals' drinking water for 5 days. At the bottom is a bar diagram with the statistical analysis from at least three independent experiments.

Article Snippet: Cell surface markers were stained using the following reagents: Fc-block (Pharmingen), anti-IgM-phycoerythrin (PE) (Dianova), anti-IgD-fluorescein isothiocyanate (FITC) (Pharmingen), anti-B220-FITC (Pharmingen), monoclonal antibody (MAb) 493-biotin ( 25 ), annexin V (Pharmingen), anti-IgM a -biotin (Pharmingen), anti-IgM b -FITC (Pharmingen), anti-Thy1.1-FITC (Pharmingen), anti-Thy1.2-PE (Pharmingen), and streptavidin-PE (Pharmingen).

Techniques: Flow Cytometry, Staining, Expressing

Journal: Leukemia

Article Title: Clonal B cells in Waldenström's macroglobulinemia exhibit functional features of chronic active B-cell receptor signaling

doi: 10.1038/leu.2016.8

Figure Lengend Snippet: BCR crosslinking elicits augmented phosphoresponses in WM cells. ( a ) anti-IgM induced fold change in phosphorylation in HD B-cells (black) and WM cells (red) expressed in the arcsinh scale (HD n =9, WM n =23, *** P <0.001, ** P <0.01, * P <0.05, Mann–Whitney test). ( b ) Dose response of anti-IgM induced fold change in pPLCγ2 in HD (black) and WM (red) cells and EC 50 values of all individuals (HD n =4, WM n =5, * P <0.05, Mann–Whitney test). ( c ) Kinetics of anti-IgM induced fold change in pPLCγ2 in HD (black) and WM (red) cells. At 0, fold change is 0 by definition. (HD n =4, WM n =6, ** P <0.01, * P <0.05, Mann–Whitney test). ( d ) Inhibition of anti-IgM induced pPLCγ2. Stimulation was performed for 4 min after a 60 min in vitro preinhibition with Dasatinib (10 μ M ), Tamatinib (10 μ M ) and Ibrutinib (10 μ M ). Here illustrated as a ratio from dimethyl sulfoxide (DMSO) treated matched control samples (WM n =5, repeated measures analysis of variance (ANOVA) with Tukey's multiple comparison test, ^ P <0.05, ^^^^ P <0.0001, ^ represents significant difference from DMSO matched control, * P <0.05, ** P <0.01, * represents significant difference in matched samples among different inhibitors). ( e ) Inhibition of anti-IgM induced pPLCγ2 at 6 months of continuous ibrutinb treatment, as a ratio from pre-ibrutinib matched anti-IgM induced pPLCγ2 levels (WM n =8, Wilcoxon test). ( f ) correlation between anti-IgM induced pPLCγ2 ratio and percentage change in intratrabecular (IT) space clonal infiltration post-ibrutinib treatment (Pearson test).

Article Snippet: Additional surface markers used included anti-human anti-IgM F(ab′) 2 —FITC (Invitrogen Biosource, Carlsbad, CA, USA), anti-IgM—Brillian Violet 650 (BD Biosciences), clone MHM-88, anti-CD22—APC, clone S-HCL-1 (BD Biosciences) and anti-CD45—APC-H7, CLONE 2D1 (BD Biosciences).

Techniques: MANN-WHITNEY, Inhibition, In Vitro

Journal: Leukemia

Article Title: Clonal B cells in Waldenström's macroglobulinemia exhibit functional features of chronic active B-cell receptor signaling

doi: 10.1038/leu.2016.8

Figure Lengend Snippet: Intra-WM signaling heterogeneity is driven by differential BCR expression and correlates with distinct clinical outcomes. ( a ) Intra-WM anti-IgM-induced phophoprotein variance (HD, black; WM, red). ( b ) Inter-phosphoprotein Spearman correlation network analysis (positive correlations, red; negative correlations, blue). ( c ) Agglomerative clustering analysis for 23 patients interrogated for all six phosphoproteins on IgM crosslinking. ( d ) Patient status on sample collection ( χ 2 test P <0.0001). ( e ) Time to second line treatment from diagnosis date to sample collection date (log-rank survival analysis). ( f ) sIgM levels among groups (HD n =4, WM high n =6, WM healthy-like n =5, ** P <0.01, analysis of variance (ANOVA) test). ( g ) Phosphatase score for pPLCγ2 among groups (HD n =9, WM high n =13, WM healthy-like n =10, ANOVA test).

Article Snippet: Additional surface markers used included anti-human anti-IgM F(ab′) 2 —FITC (Invitrogen Biosource, Carlsbad, CA, USA), anti-IgM—Brillian Violet 650 (BD Biosciences), clone MHM-88, anti-CD22—APC, clone S-HCL-1 (BD Biosciences) and anti-CD45—APC-H7, CLONE 2D1 (BD Biosciences).

Techniques: Expressing

Journal: Nature Communications

Article Title: Differential organization of tonic and chronic B cell antigen receptors in the plasma membrane

doi: 10.1038/s41467-019-08677-1

Figure Lengend Snippet: Arrangement of mIgM-containing BCRs in the plasma membrane of human B cells. a Schematic depiction of the staining procedure and the generation of plasma membrane sheets. b , e , h Images of membrane sheets of unstimulated Ramos B cells ( b ), unstimulated primary B cells ( e ), and Ramos cells stimulated with anti-IgM F(ab’) fragments for 30 min on ice ( h ). Membrane sheets were stained with the membrane marker R18 (confocal images pseudo-colored in green) and anti-human IgM affibody-Star635P (STED images displayed in red). White squares on central images indicate the zoomed regions that are displayed in the images to their right. Scale bars represent 5 µm and 1 µm for the low and high zoom images respectively. c , f , i Example histograms showing the fluorescence intensity distributions of single Star635P-conjugated affibodies on coverslips (black curves, control) and mIgM-spots present in membrane sheets (red curves, mIgM-BCRs). d , g , j Percentages of different mIgM-BCR arrangements in plasma membrane sheets were calculated after pooling the spot intensities of dozens of membrane sheets together and then fitting the intensity distribution to a sum of Gaussian curves (Supplementary Fig. ). Data were obtained from four and three independent experiments including a total of 30 membranes from unstimulated and 15 membranes from F(ab’) -stimulated Ramos B cells, respectively. The experiments with primary human B cells were performed three independent times from two different donors and 30 membrane sheets were analyzed in total. Error bars represent the 68% confidence interval, corresponding to 1 standard deviation. Histogram’s source data are provided as a Source Data file

Article Snippet: The anti-IgM affibody K d was measured with a NanoTemper Microscale Thermophoresis Monolith NT.115Pico (NanoTemper Technologies GmbH, Munich, Germany).

Techniques: Staining, Marker, Fluorescence, Standard Deviation

Journal: Nature Communications

Article Title: Differential organization of tonic and chronic B cell antigen receptors in the plasma membrane

doi: 10.1038/s41467-019-08677-1

Figure Lengend Snippet: mIgM-BCRs do not arrange in large protein islands at the plasma membrane. a Schematic depiction of the TIRF/dSTORM setup imaging depth. b Representative TIRF/dSTORM image of an intact Ramos B cell stained with CF TM 647-conjugated anti-IgM affibodies. The dotted white trace delineates the area of the plasma membrane close to the glass coverslip imaged under TIRF/dSTORM mode. The white square indicates the zoomed area shown in the image to the right. Scale bars represent 1 µm and 0.5 µm for the low and high zoom, respectively. c Scatter plot displaying the spot size (FWHM) distribution unstimulated Ramos cells and as resolution reference the scatter plot of single CF TM 647- affibodies spread on a glass coverslip

Article Snippet: The anti-IgM affibody K d was measured with a NanoTemper Microscale Thermophoresis Monolith NT.115Pico (NanoTemper Technologies GmbH, Munich, Germany).

Techniques: Imaging, Staining